Berberine Bridge Enzyme (BBE) is a flavin-containing oxidase that catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, both intermediates in the benzylisoquinoline alkaloid biosynthesis pathways in plants. Alkaloids belonging to this family, including berberine, palmatine, and sanguinarine, have confirmed antimicrobial and anticancer activities. The regioselective cyclization reaction catalyzed by BBE is novel; only two other flavoenzymes have been shown to catalyze similar reactions, and this cyclization has not yet been replicated by synthetic organic methods. The goal of this proposed project is mechanistic analysis of the enzyme activity, including differentiation between stepwise and concerted mechanisms, and determination of the roles of active site residues. These studies will involve steady-state and rapid-reaction kinetics, as well as primary and solvent kinetic isotope effects, in order to determine individual rate constants associated with the BBE-catalyzed reaction and to identify rate-determining steps. The roles of active site residues will be probed by site-directed mutagenesis studies.